The three ‘P’s of mitophagy: PARKIN,PINK1, and post-translational modifications

Two Parkinson''s disease (PD)-associated proteins, the mitochondrial kinase PINK1 and the E3-ubiquitin (Ub) ligase PARKIN, are central to mitochondrial quality control. In this pathway, PINK1 accumulates on defective mitochondria, eliciting the translocation of PARKIN from the cytosol to mediate the clearance of damaged mitochondria via autophagy (mitophagy). Throughout the different stages of mitophagy, post-translational modifications (PTMs) are critical for the regulation of PINK1 and PARKIN activity and function. Indeed, activation and recruitment of PARKIN onto damaged mitochondria involves PINK1-mediated phosphorylation of both PARKIN and Ub. Through a stepwise cascade, PARKIN is converted from an autoinhibited enzyme into an active phospho-Ub-dependent E3 ligase. Upon activation, PARKIN ubiquitinates itself in concert with many different mitochondrial substrates. The Ub conjugates attached to these substrates can in turn be phosphorylated by PINK1, which triggers further cycles of PARKIN recruitment and activation. This feed-forward amplification loop regulates both PARKIN activity and mitophagy. However, the precise steps and sequence of PTMs in this cascade are only now being uncovered. For instance, the Ub conjugates assembled by PARKIN consist predominantly of noncanonical K6-linked Ub chains. Moreover, these modifications are reversible and can be disassembled by deubiquitinating enzymes (DUBs), including Ub-specific protease 8 (USP8), USP15, and USP30. However, PINK1-mediated phosphorylation of Ub can impede the activity of these DUBs, adding a new layer of complexity to the regulation of PARKIN-mediated mitophagy by PTMs. It is therefore evident that further insight into how PTMs regulate the PINK1–PARKIN pathway will be critical for our understanding of mitochondrial quality control.     

PRAS40活性调节与功能的研究进展

PRAS40是蛋白激酶B(PKB/Akt)的作用底物,亦是m TORC1的特异性结合蛋白,有多个位点可发生磷酸化,其中Thr246磷酸化受Akt调控,而Ser183、Ser212及Ser221等磷酸化主要受m TORC1调控。磷酸化修饰的PRAS40可调节与Raptor及14-3-3等蛋白的结合,参与Akt、m TORC1活性的调控。PRAS40具有调控细胞增殖、参与神经损伤保护等作用,在胰岛素抵抗、神经退行性病变及肿瘤中扮演重要角色,有望成为药物作用的新靶点。     

Molecular diagnosis of coenzyme Q10 deficiency: an update

Introduction: Coenzyme Q₁₀ (CoQ) deficiency syndromes comprise a growing number of genetic disorders. While primary CoQ deficiency syndromes are rare diseases, secondary deficiencies have been related to both genetic and environmental conditions, which are the main causes of biochemical CoQ deficiency. The diagnosis is the essential first step for planning future treatment strategies, as the potential treatability of CoQ deficiency is the most critical issue for the patients.

Areas covered: While the quickest and most effective tool to define a CoQ-deficient status is its biochemical determination in biological fluids or tissues, this quantification does not provide a definite diagnosis of a CoQ-deficient status nor insight about the genetic etiology of the disease. The different laboratory tests to check for CoQ deficiency are evaluated in order to choose the best diagnostic pathway for the patient.

Expert commentary: New insights are being discovered about the implication of new proteins in the intricate CoQ biosynthetic pathway. These insights reinforce the idea that next generation sequencing diagnostic strategies are the unique alternative in terms of rapid and accurate molecular diagnosis of CoQ deficiency.     

Different mechanisms of homologous and heterologous μ-opioid receptor phosphorylation

The efficiency of μ-opioid receptor signalling is tightly regulated and ultimately limited by the coordinated phosphorylation of intracellular serine and threonine residues. Here, we review and discuss recent progress in the generation and application of phosphosite-specific μ-opioid receptor antibodies, which have proved to be excellent tools for monitoring the spatial and temporal dynamics of receptor phosphorylation and dephosphorylation. Agonist-induced phosphorylation of μ-opioid receptors occurs at a conserved 10 residue sequence ³⁷⁰TREHPSTANT³⁷⁹ in the receptor''s carboxyl-terminal cytoplasmic tail. Diverse opioids induce receptor phosphorylation at S375, present in the middle of this sequence, but only high-efficacy opioids have the ability to drive higher order phosphorylation on flanking residues (T370, T376 and T379). S375 is the initiating residue in a hierarchical phosphorylation cascade. In contrast, agonist-independent heterologous μ-opioid receptor phosphorylation occurs primarily at T370. The combination of phosphosite-specific antibodies and siRNA knockdown screening also facilitated the identification of relevant kinases and phosphatases. In fact, morphine induces a selective S375 phosphorylation that is predominantly catalysed by GPCR kinase 5 (GRK5), whereas multisite phosphorylation induced by high-efficacy opioids specifically requires GRK2/3. By contrast, T370 phosphorylation stimulated by phorbol esters or heterologous activation of G_q-coupled receptors is mediated by PKCα. Rapid μ-opioid receptor dephosphorylation occurs at or near the plasma membrane and is catalysed by protein phosphatase 1γ (PP1γ). These findings suggest that there are distinct phosphorylation motifs for homologous and heterologous regulation of μ-opioid receptor phosphorylation. However, it remains to be seen to what extent different μ-opioid receptor phosphorylation patterns contribute to the development of tolerance and dependence in vivo.

LINKED ARTICLES

This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2     

Prolongation of tension on reoxygenation following myocardial hypoxia: a possible role for mitochondria in muscle relaxation.

The mechanism for tension prolongation during reoxygenation following myocardial hypoxia was investigated. It was found that prior addition of isoproterenol, reserpine, quinidine, lidocaine and insulin or a change in pH, temperature, stimulation rate, preload or duration of hypoxia did not qualitatively alter the appearance of the phenomenon. On reoxygenating hypoxic preparations in the presence of 5, 10 or 20% oxygen, tension prolongation was clearly present when little increase in isometric tension was evident. Inhibition of glycolysis by iodoacetate did not alter the appearance of the phenomenon during reoxygenation. Three respiratory inhibitors, antimycin A, rotenone and cyanide, at concentrations which did not prevent an increase in isometric tension during recovery from myocardial hypoxia, all completely prevented the appearance of tension prolongation. Two uncouplers of oxidative phosphorylation, 2–4 dinitrophenol and carbonyl cyanide m-chlorophenyl hydrazone at concentrations large enough to lead to mechanical deterioration despite the presence of oxygen, failed to prevent the appearance of tension prolongation upon reoxygenation. It is concluded that myocardial respiratory activity, independent of ability to generate high energy phosphate, appears capable of altering the duration of mechanical events during reoxygenation of hypoxic heart muscle.     

Dicarbonyl Electrophiles Mediate Inflammation-Induced Gastrointestinal Carcinogenesis

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